Complete chloroplast genomes of two mint species (Lamiaceae) from Al-Madinah, Saudi Arabia: phylogenetic and genomic comparative analyses

Abstract The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. However, the systematics of the genus Mentha is very complicated and still uncertain. This is largely because of the presence of frequent interspecific hybridization, variation in chromosome number, cytomixis, polymorphism in morphology and essential oil composition under different environmental conditions, colonial mutant propagation, as well as the occurrence of polyploidy, aneuploidy, and nothomorphs. Here, we present the plastome assemblies for a wilt-resistant Saudi Arabian accession of Mentha longifolia (L.) Huds and an alien hybrid Mentha × verticillata L. which are 152,078 bp and 152,026 bp in length, respectively, and exhibited large single-copy (LSC) and small single-copy (SSC) regions separated by a pair of inverted repeat regions. The chloroplast genome of M. longifolia has 133 annotated genes, including 88 protein-coding genes and 37 tRNAs while M. × verticillata has 133 annotated genes, including 87 protein-coding genes and 38 tRNAs. Both cp genomes have eight rRNA genes. Phylogenetic analysis using a total chloroplast genome DNA sequence of 17 species revealed that M. longifolia sequenced in this study did not form a sister relationship with M. longifolia from another study. This opens a window for further investigations.

The genus Mentha L. (mint) is one of the most significant taxa of the Lamiaceae, and it comprises 24 species, among which 15 are hybrids (POWO). Species in the genus are almost exclusively perennial, herbaceous plants, which are cosmopolitan in distribution. The genus is represented by three species in Saudi Arabia: Mentha longifolia (L.) Huds 1762 ( (Collenette 1985;POWO 2022). All the species are used in herbal teas, alone or as spice mixtures for many foods to offer aroma and flavor. In addition, Mentha species had been used for the treatment of many diseases such as throat infection, bronchitis, nausea, and ulcerative colitis (Ahmed et al. 2015;Vining et al. 2017). This makes many Mentha species very valuable for industry, as mint oil is a supplement for wide spectrum of products. Other uses of mint essential oils include antipruritic, astringent, antiseptic, and antimicrobial activities, and for treating neuralgia, myalgia, headaches, and migraines (Ahmed et al. 2015;Jedrzejczyk and Rewers 2018).
Despite the significance of the species of the genus Mentha, identification and phylogenetic relationships among the species are very complicated and still uncertain. This is largely as a result of the variation in basic chromosome number, frequent interspecific hybridization, cytomixis, polymorphism in morphology and essential oil composition under different environmental conditions, colonial mutant propagation, as well as the occurrence of polyploidy, aneuploidy, and nothomorphs (Gobert et al. 2002;Tucker and Chambers 2002;Denslow and Poindexter 2009;Jabeen et al. 2012;Tucker 2012;Jedrzejczyk and Rewers 2018). This is the cause of the existence of numerous synonymous names (over 3000 published names in the genus), and still uncertain taxonomy of the genus. To solve this problem, there is need to sequence and characterize the plastome of the species.
Two sampled specimens were identified by Dr. Faten Z. Filimban and deposited in the Herbarium of King Abdulaziz University in Saudi Arabia (KAU) (contact info: malgandaby@ yahoo.com) including M. longifolia (voucher no. FK1021) and M. Â verticillata (voucher no. FK1022) all from Wadi Aqiq near Dhul Hulaifah area, Al-Madinah Province, Saudi Arabia (24.4233720 N, 39.5621330 E and 24.42398 N, 39.56723 E, respectively). The plants were collected in non-protected areas; hence no permission is required for the collection of the species. Total genomic DNA was extracted using plant DNA (BioTeke Corporation, Beijing, China). Genome sequencing was performed on an Illumina HiSeq 2500 platform (Illumina Inc., San Diego, CA). Approximately, 6.0 and 5.1 GB of clean data were obtained for M. longifolia and M. Â verticillata, respectively, and assembled using NOVOPlasty (Dierckxsens et al. 2017). The complete chloroplast genome of Mentha longifolia (NC_032054.1) was used as seed and reference. The orientation of the assembled sequences was further corrected manually using Geneious prime 2020.0.5 (Biomatters Ltd., Auckland, New Zealand).
The complete cp genome was annotated with the online annotation tool GeSeq and annotations were corrected manually with the sequin v. 15. 50 (Biomatters Ltd., Auckland, New Zealand).
The circular chloroplast genome of M. longifolia was 152,078 bp (GenBank accession no. ON124917) in size, contained a large single-copy (LSC) region of 83,220 bp and a small single-copy (SSC) region of 17,652 bp, and separated by a pair of inverted repeats (IRs) regions of 25,603 bp. The cp genome has 133 annotated genes, including 88 protein coding genes, 37 tRNA genes, and eight rRNA genes. The base compositions of the chloroplast genome were uneven (31.5% A, 19.2% C, 18.6% G, and 31.5% T), with an overall GC content of 37.8%.
The complete chloroplast genome of M. Â verticillata was 152,026 bp in length (GenBank accession no. ON124918) and composed of IRs of 25,605 bp which divide LSC of 83,165 bp and SSC of 17,655 bp, the average GC content was 37.8% (30.7% A, 19.2% C, 18.6% G, and 31.5% T). There are 133 genes annotated, including 87 protein-coding genes, 38 tRNA genes, and eight rRNA genes.
To identify the phylogenetic positions of M. longifolia and M. Â verticillata among the species Nepetoideae, we downloaded 13 published complete chloroplast genome sequences of Nepetoideae and chloroplast genome of Teucrium mascatense (Ajugoideae), Congea tomentosa (Symphorematoideae), and Dicrastylis parvifolia (Prostantheroideae) to be used as outgroup to construct the phylogenetic tree. The plastome sequences were aligned with MAFFT v.7 (Katoh and Standley 2013), and analyzed using Bayesian's inference analysis. The result clearly showed that the tribe is monophyletic based on the samples of species and also supported the division of Salviinae, Nepetinae, and Menthinae subtribes with strong support with posterior probability (PP)¼1.0 (Figure 1). The phylogenetic tree revealed that M. Â verticillata is a sister to Mentha spicata whereas M. longifolia formed a sister relationship with a clade of M. canadensis and M. Â piperita. All these relationships were strongly supported (PP ¼ 1.0). Interestingly, M. longifolia sequenced in this study did not form a sister relationship with M. longifolia from South Africa, this might be as a result of phylogeographic factors. This opens a window for further investigations.
Author contributions SSY, FZB, and AB were involved in the conception and design; HC and SSY performed the analysis; AB and HC drafted the paper; FZB and SSY revised it critically for intellectual content; all authors approved the final version to be published and agreed to be accountable for all aspects of the work. All the authors meet the criteria for authorship as per the ICMJE criteria.

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